Field of the Invention
Hypersensitive individuals undergo an "altered state" as a result of contact with the antigens from an allergen, leading to the formation of antibodies thereto. Subsequent contact with one of those antigens or a structurally similar substance can evoke in an allergic individual a pathological reaction, due to the presence of such antibodies. When these individuals inhale or ingest the offending antigen, a prominent and common manifestation includes hay fever, asthma or hives. These antibodies are tissue-sensitizing and can be referred to as reagin Ig. There are five classes of antibody; IgE, IgG, IgA, IgM and IgD. IgE constitutes the class of antibodies of major importance in atopic allergy.
In vivo diagnostic tests for atopy, i.e. to determine the presence of IgE produced in response to allergens, are generally conducted by injecting small amounts of the suspected allergen into the skin of the sensitive individual, and examining the injected site for formation on the skin of an irregular wheal surrounded by a zone of erythema. In addition to in vivo tests, in vitro diagnostic tests have been developed. One type of in vitro diagnostic test involves analytical procedures which can be broadly classified as "specific binding assays".
Specific binding assay procedures, with reference to in vitro diagnostic tests for atopy, involve the detection of a ligand in a body fluid. A typical specific binding assay procedure is the determination of IgE antibodies (ligand) in the blood serum of a hypersensitive individual. U.S. Pat. No. 3,720,760 is directed to an in vitro diagnostic test; the technique disclosed by the patentee is commonly referred to as the "RAST" (radioallergosorbent test).
In order to illustrate the scope of the present invention, the RAST technique is decribed herewith in detail. The test involves a first step of attaching a test allergen (antigen) to an insoluble polymeric substrate to form a bound solid-phase antigen. The second step involves contacting the solid-phase antigen with serum from an atopic individual (having IgE antibody present); the IgE is the ligand referred to earlier. The IgE attaches to IgE receptor sites on the antigen, to form an antigen-ligand complex. The third step involves contacting the complex with a labelled constituent, which is a conjugate of a labelling substance and a binding component (capable of binding to the ligand), e.g., radio-labelled anti-IgE.
Various labelling substances can be used. Because of the hazard and difficulty of handling radioactive materials, many new assay systems, which can be designated as "RAST-type" systems, have been devised using materials other than radioactive isotopes as the labelling substance. Examples of labelling substances include free radicals, fluorescent molecules (e.g., fluoroscein isothiocyanate and rhodamine dyes), luminescent molecules, bacteriophages, enzymes, coenzymes and enzyme inhibitors.
Antibodies are normally composed of four chains, two light chains and two heavy chains. In both types of chains there is a constant region and variable region, i.e., the amino acid sequence of the region is relatively constant or varible, respectively. The amino acid sequence of the variable region depends upon the identity of the antigen against which it is directed; the variable region of the antibody molecule that binds the antigen is included within the antibody binding fragment (Fab) obtained by papain digestion. The constant region does not vary with regard to the antigen against which the antibody molecule is directed but is characteristic for a given antibody class. Part of the heavy chain constant region forms the other fragment (Fc) released by papain digestion.
The IgE binds to the antigen via the antibody fragment (Fab) region of the antigen molecule, leaving the Fc region exposed. The radiolabelled anti-IgE then binds to the IgE via the Fc region of the IgE.
The RAST reaction product, is a substrate having attached thereto three "layers": (1) solid-phase antigen; (2) IgE specifically directed against the antigen; and (3) radiolabelled anti-IgE which specifically reacts with IgE. The radiation emitted by the radiolabelled reaction product is a measure of the amount of antigen specific IgE present in the test serum. The use of the RAST described above enables determination of the presence and, to a limited extent, the quantity of IgE present in a patient's serum, capable of reacting with the antigen attached to the substrate. This test in conjunction with skin testing gives an indication as to whether or not the patient is allergic to the antigen.
Following diagnosis of atopy, a common method of treating allergic disorders is by immunizing ("desensitizing") the individual with a course of injections of gradually increasing amounts of allergen extracts. Because of the danger of triggering anaphylactic shock, the amounts of allergen extract administered must be carefully controlled.
Variation in the potency of the allergen extract administered, as indicated by measurement of the reactivity in the skin of sensitive individuals, has been observed. This variation of potency indicates the desirability of improving the standardization of allergen extracts. Difficulties with standardization of allergen extracts is thought to arise because allergen (e.g. pollen) extracts, are usually complex mixtures, and it has been difficult to determine which constituents are responsible for eliciting either an allergic reaction or therapeutic effect during therapy. Methods of expressing potency have been largely indirect and based either on a simple ratio of raw source materials to volume of extracting fluid or on the protein nitrogen of the extract. Since most allergens appear to be proteinaceous in nature, designation of potency by protein nitrogen units (PNU), defined as 0.01 .mu.g of phosphotungstic acid-precipitable nitrogen, has gained wide acceptance. PNU content however, is not necessarily related to biologic activity, because allergen extracts may contain proteins which are not allergenically active. Furthermore, specific protein allergens may be denatured during extraction or storage, with loss of their biologic activity, yet still appear as protein in a chemical determination.
A second use of binding assay techniques, such as the RAST, is in the measurement of the potency of allergens, in an attempt to standardize allergens for use in diagnostic skin testing and for in vivo desensitization. This utilization is carried out by maintaining the second and third layers (i.e., IgE and labelled anti-IgE respectively) constant, and adding increasing amounts of soluble antigen in the first step. The principle of the technique, is based on a competition between solid and fluid-phase antigens for IgE receptor sites in the first step of the RAST. As the amount of soluble antigen is increased, a smaller amount of IgE complexes with the solid-phase antigen because of the increase in complexes formed between the fluid-phase antigen and IgE. This in turn decreases the percent binding of radiolabelled anti-IgE to solid-phase antigen in the third step of the RAST. This decrease in percent binding of anti-IgE to solid phase antigen produces a decrease in the amount of radiation emitted by the solid-phase.
Starting with 100 percent binding, by careful titration, the quantity of soluble antigen needed to achieve a 50 percent inhibition of the binding can be determined. By plotting the amount of soluble antigen required, against the amount of inhibition produced, on semilog paper, an approximate linear relationship can be established. In particular, between the region of 30 to 70 percent inhibition the semilog plots are reasonably linear. The end point in these titrations can be arbitrarily selected as the amount of allergen extract required for 50 percent inhibition. In theory, comparison of a series of allergen extracts would provide comparative data sufficient to enable standardization of the extracts. [See J. Allergy Clin. Immunol. 53; 158-159 (1974)]. As described below, this method suffers from various disadvantages.
One disadvantage lies in the anti-IgE itself. The final reagent used in the RAST, the radiolabelled anti-IgE, is produced by radiolabelling with a radioactive iodide salt. This produces radiolabelled anti-IgE with a half-life sufficiently short to achieve the necessary sensitivity for the measurement of low concentrations of IgE. However, the short half-life means that the radiolabelled anti-IgE is changing rapidly and prevents its use as a standard reference reagent over any reasonable time period. In addition, harsh iodination conditions and subsequent radiation damage during the radiolabelling process produce variations in the radiolabelled anti-IgE. Because of these variations, it is not possible to directly compare two assays performed at different times, using the same batch of radiolabelled anti-IgE or at the same time using different batches of radiolabelled anti-IgE. By standardizing the radiolabelled anti-IgE according to the present invention, these disadvantagesare obviated.
Standardization by RAST inhibition suffers other disadvantages. The RAST itself is not a standardized technique. Typically, radioimmunoassays are standardized by the establishment of a stated well-defined reference reagent, either an antigen or antibody. However, allergens are complex mixtures which are not-well defined and their stability is not clearly known. Thus at best this method can only partially standardize the RAST. However, it has been proposed (See J. Allergy Clin. Immunol. 53: 158-169, 1974) that serum from atopic individuals could be used as a reference reagent for the more common allergies. This too would only result in a partial standardization of the RAST because serum represents a complex mixture with ill-defined antibody specificities. In spite of the inherent difficulties, ranking of atopic sera for use in the direct RAST has been proposed (See Advances in Diagnosis of Allergy: RAST pp. 85-99 Symposium specialists [1975]).
In theory, radiolabelled anti-IgE for the third step of RAST could be standardized by keeping the first and second layers constant and comparing the radiolabelled anti-IgE in the third layer. The standardized radiolabelled anti-IgE could then be used to standardize the RAST test itself. Standardization of anti-IgE RAST with standardized radiolabelled anti-IgE would be useful in that one standard reagent could be used, regardless of the allergen extract present in the first layer. However, because the reagents used in the first two steps of RAST are complex mixtures of ill defined stability, these steps are difficult to reproduce precisely from one experiment to the next. Since it is also difficult to define the activity of samples of radiolabelled anti-IgE precisely, this reagent could not easily be used as a reference standard.
As referred to earlier, an important example of a binding assay employing a labelled compound is radioimmunoassay, such as radioallergosorbent (RAST) techniques. Alternative labelled materials suggested are free radicals, fluorescent molecules, luminescent molecules, bacteriophages, enzymes, coenzymes or enzyme inhibitors. Similar difficulties exist for defining the activity of any labelled material, irrespective of the material against which it is directed or the type of labelled compound used.